Btnl9 and ermap as novel inhibitors of the immune system for immunotherapies

ABSTRACT

Provided are methods of treating a tumor in a subject with a BTNL9-binding antibody. Also provided are methods of treating a tumor in a subject with an ERMAP-binding antibody. A fusion protein comprising a BTNL9 or ERMAP and related compositions and encoding nucleic acids are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/084,124, filed Nov. 25, 2014, the contents of which are herebyincorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersDK083076 and DK007218 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The disclosures of all publications, patents, patent applicationpublications and books referred to herein, are hereby incorporated byreference in their entirety into the subject application to more fullydescribe the art to which the subject invention pertains.

Cancers, autoimmune diseases, infectious diseases, and transplantationrejection are serious public health problems in the US and othercountries. The immune system, particularly T cells, plays critical rolesin these diseases.

The B7 ligand family and CD28 receptor family control T cell activationand function. Both CTLA-4 and PD-1 are members of the CD28 family, andan antibody against CTLA-4 (Yervoy from Bristol-Myers Squibb) and anantibody against PD-1 (Keytruda, Merck) were approved by the FDA as newdrugs for melanoma in 2011 and 2014, respectively. PD-L1 is a member ofthe B7 family, and antibodies to PD-L1 are in clinical trials withcancer patients. In additional, CTLA-4-Ig (Orencia) was approved by theFDA as a new drug for adult rheumatoid arthritis.

The existing technologies work by blockade of the B7/CD28 familymembers. The butyrophilin family is related to the B7 family, but theirexpression and functions in the immune system are largely unknown.

The present invention provides addresses the need for improved therapiesand therapeutics based on targeting BTNL9 or ERMAP.

SUMMARY OF THE INVENTION

A method of treating a tumor in a subject comprising administering tothe subject an amount of a BTNL9-binding antibody, or BTNL9-bindingfragment thereof, sufficient to inhibit a BTNL9 and treat the tumor.

A method of treating a tumor in a subject comprising administering tothe subject an amount of a ERMAP-binding antibody, or ERMAP-bindingfragment thereof, sufficient to inhibit a ERMAP and treat the tumor.

A method of treating a tumor in a subject comprising administering tothe subject an amount of a BTNL2-binding antibody, or BTNL2-bindingfragment thereof, sufficient to inhibit a BTNL2 and treat the tumor.

A method of treating an autoimmune disease in a subject comprisingadministering to the subject an amount of an isolated, plasma-solubleBTNL9 to treat the autoimmune disease.

A method of treating an autoimmune disease in a subject comprisingadministering to the subject an amount of an isolated, plasma-solubleERMAP to treat the autoimmune disease.

A method of treating an autoimmune disease in a subject comprisingadministering to the subject an amount of an isolated, plasma-solubleBTNL2 to treat the autoimmune disease.

An isolated, recombinant fusion polypeptide comprising a BTNL9 fused toan immunoglobulin polypeptide.

An isolated, recombinant fusion polypeptide comprising an ERMAP fused toan immunoglobulin polypeptide.

An isolated, recombinant fusion polypeptide comprising a BTNL2 fused toan immunoglobulin polypeptide.

An isolated chimeric nucleic acid encoding an isolated recombinantfusion polypeptide as described herein.

A composition comprising an isolated recombinant fusion polypeptide asdescribed herein and a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. mRNA transcripts of BTN and BTNL family members in mousetissues and antigen-presenting cells relative to the reference geneOAZ1. (A) Transcripts in naïve mouse tissues. Transcripts of BTN familymembers (upper panel) show that BTN transcripts are most abundant inmammary tissue. Transcripts of BTNL family members with intracellularSPRY domains (center panel) and of those without intracellular SPRYdomains (bottom panel) are highest in the small and large intestines.Transcripts are also generally detectable in other tissues, particularlythe primary lymphoid organs; MOG transcripts are highest in braintissue. SP, spleen; LN, pooled inguinal and brachial lymph nodes; TH,thymus; BM, bone marrow; MLN, mesenteric lymph node; BR, brain; LU,lung; HE, heart; LI, liver; ST, stomach; SM, small intestine; LG, largeintestine; PA, pancreas; KI, kidney; BL, bladder; SV, seminal vesicles;PR, prostate; EP, epididymis; TE, testis; FT, Fallopian tube; VA,vagina; UT, uterus; OV, ovary; MA, mammary gland. (B) Transcripts inresting and activated antigen-presenting cells (APCs). Transcripts formost genes are detectable in all three professional APCs, are in mostcases lower in macrophages compared to dendritic cells and B cells, andare generally not strongly up- or down-regulated upon activation by LPSor PMA+ionomycin.

FIG. 2A-2C. Most recombinant BTN and BTNL family members inhibit thefunction of anti-CD3-activated primary mouse T cells. T cells wereisolated from mouse spleen and lymph nodes by magnetic cell separationand activated with 2.5 μg/mL plate-bound anti-CD3 in the presence of 4μg/mL plate-bound BTN- and BTNL-human Ig recombinant fusion proteins.(A) All BTN and BTNL fusion proteins, with the exception of ERMAP andMOG, reduce the metabolic activity of CD4+ T cells by 3 dayspost-activation. Human Ig (hIg), B7x-hIg, and B7.2-hIg fusion proteinsare included as baseline, negative, and positive controls foractivation, respectively. (B) BTN and BTNL fusion proteins reduceproliferation of activated CD4+ and CD8+ T cells. CD90.2+ T-cells werepulsed with CFSE prior to activation for 4 days, and stained withfluorescence-labeled antibodies to CD4 and CD8. CD4+ T cells (upperpanel) showed reduced proliferation in the presence of BTN, BTN2, BTNL1,BTNL2 (full- and partial length), BTNL4, BTNL6, BTNL9, and ERMAP, andCD8+ T cells (panel) showed reduced proliferation in the presence ofBTN, BTN2, BTNL1, BTNL2 (full- and partial-length), BTNL4, BTNL6, andBTNL9. The division indices, i.e., average number of divisions (closedbars), and percent divided (open bars) of CD4+ and CD8+ T cells areshown to the right of the histograms. (C) Secretion of the cytokinesIFN-γ, IL-2, TNF-α, and IL-17A by CD4+ T cells 2 days post-activation isreduced in the presence of BTN and BTNL fusion proteins compared tobaseline (hIg).

FIG. 3A-3B. CD4+ (A) and CD8+ (B) T cells activated for 3 days express areceptor for BTN and BTNL proteins. Total lymph node cells wereactivated by plate-bound 2.5 μg/mL anti-CD3 and incubated withbiotinylated BTN-, BTNL-hIg, or hIg-fusion protein, stained withfluorescence-labeled antibodies to CD4 and CD8, and evaluated for fusionprotein binding with fluorescence-labeled streptavidin. Both activated Tcell subsets express a receptor for all BTN and BTNL family members,with the exception of MOG, which is consistent with functional data.

FIG. 4. The IgV1 domain of human BTNL2 bound human CD8 T cells, human Bcells, and human NK cells. BTNL2-IgV1-Ig protein (open histograms) andcontrol Ig (shaded histograms) are shown in FACS assays.

FIG. 5. The IgV1 domain of human BTNL9 bound human CD8 T cells, human Bcells, and human NK cells. BTNL9-IgV1-Ig protein (open histograms) andcontrol Ig (shaded histograms) are shown in FACS assays.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the first disclosure that butyrophilin-like 9(BTNL9) and erythroblast membrane-associated protein (ERMAP), twomembers of the butyrophilin family, inhibit T cell functions. A shortform of BTNL2 was also strongly inhibitory. In particular, evidence thatrecombinant proteins of BTNL9 and ERMAP inhibit cell proliferation andcytokine production of T cells and that activated T cells have receptorsfor BTNL9 and ERMAP is provided. Since BTNL9 and ERMAP are expressed byimmune cells or other cells. Applications of targeting these twomolecules to enhance immunity (e.g. cancer immunotherapy) or decreaseimmunity (e.g. therapy of autoimmune diseases) are encompassed. Also,applications of using BTNL2, especially the short or “partial” form areencompassed.

BTNL9 is an inhibitor of the immune system. Therefore blockade ofBTNL9-mediated immune suppression with blockers (e.g. monoclonalantibodies to BTNL9) can be used for treatment of human cancers andinfectious diseases, while enhancement of BTNL9-mediated immunesuppression with soluble proteins (e.g. BTNL9-Ig) can be used fortreatment of autoimmune diseases and transplantation.

ERMAP is an inhibitor of the immune system. Therefore blockade ofERMAP-mediated immune suppression with blockers (e.g. monoclonalantibodies to ERMAP) can be used for treatment of human cancers andinfectious diseases, and enhancement of ERMAP-mediated immunesuppression with soluble proteins (e.g. ERMAP-Ig) can be used fortreatment of autoimmune diseases and transplantation.

A method of treating a tumor is provided in a subject comprisingadministering to the subject an amount of a BTNL9-binding antibody, orBTNL9-binding fragment thereof, sufficient to inhibit a BTNL9 and treatthe tumor.

Also provided is a method of treating a tumor in a subject comprisingadministering to the subject an amount of a ERMAP-binding antibody, orERMAP-binding fragment thereof, sufficient to inhibit a ERMAP and treatthe tumor.

Also provided is a method of treating a tumor in a subject comprisingadministering to the subject an amount of a BTNL2-binding antibody, orBTNL2-binding fragment thereof, sufficient to inhibit a BTNL2 and treatthe tumor.

In an embodiment, the BTNL9 is a human BTNL9. In an embodiment, theERMAP is a human ERMAP. In an embodiment, the BTNL2 is a human BTNL2.

In an embodiment of the methods, the tumor is a tumor of a breast, lung,thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate,kidney, esophagus, or is a hematological tumor, or wherein the tumor isa lymphoid organ tumor.

In an embodiment of the methods, the antibody is administered as anadjunct to an additional anti-cancer therapy for the tumor.

In an embodiment of the methods, the amount of a BTNL9-binding antibodyis administered. In an embodiment of the methods, the BTNL9-bindingantibody binds an IgV1 domain of human BTNL9.

In an embodiment of the methods, the amount of a ERMAP-binding antibodyis administered.

In an embodiment of the methods, the amount of a BTNL2-binding antibodyis administered. In an embodiment of the methods, the BTNL2-bindingantibody binds an IgV1 domain of human BTNL2.

In an embodiment of the methods, the fragment of the antibody isadministered.

In an embodiment of the methods, the antibody is a monoclonal antibody.

In an embodiment of the invention, the BTNL9 is human BTNL9. In anembodiment human BTNL9 protein has the sequence:

(SEQ ID NO: 1) MVDLSVSPDSLKPVSLTSSLVFLMHLLLLQPGEPSSEVKVLGPEYPILALVGEEVEFPCHLWPQLDAQQMEIRWFRSQTFNVVHLYQEQQELPGRQMPAFRNRTKLVKDDIAYGSVVLQLHSIIPSDKGTYGCRFHSDNFSGEALWELEVAGLGSDPHLSLEGFKEGGIQLRLRSSGWYPKPKVQWRDHQGQCLPPEFEAIVWDAQDLFSLETSVVVRAGALSNVSVSIQNLLLSQKKELVVQIADVFVPGASAWKSAFVATLPLLLVLAALALGVLRKQRRSREKLRKQAEKRQEKLTAELEKLQTELDWRRAEGQAEWRAAQKYAVDVTLDPASAHPSLEVSEDGKSVSSRGAPPGPAPGHPQRFSEQTCALSLERFSAGRHYWEVHVGRRSRWFLGACLAAVPRAGPARLSPAAGYWVLGLWNGCEYFVLAPHRVALTLRVPPRRLGVFLDYEAGELSFFNVSDGSHIFTFHDTFSGALCAYFRPRAHDGGEHPDPLTICPLPVRGTGVPEENDSDTWLQPYEPA DPALDWW.

In an embodiment of the invention, the ERMAP is human ERMAP. In anembodiment human ERMAP protein has the sequence:

(SEQ ID NO: 2) MEMASSAGSWLSGCLIPLVFLRLSVHVSGHAGDAGKFHVALLGGTAELLCPLSLWPGTVPKEVRWLRSPFPQRSQAVHIFRDGKDQDEDLMPEYKGRTVLVRDAQEGSVTLQILDVRLEDQGSYRCLIQVGNLSKEDTVILQVAAPSVGSLSPSAVALAVILPVLVLLIMVCLCLIWKQRRAKEKLLYEHVTEVDNLLSDHAKEKGKLHKAVKKLRSELKLKRAAANSGWRRARLHFVAVTLDPDTAHPKLILSEDQRCVRLGDRRQPVPDNPQRFDFVVSILGSEYFTTGCHYWEVYVGDKTKWILGVCSESVSRKGKVTASPANGHWLLRQSRGNEYEALTSPQTSFRLKEPPRCVGIFLDYEAGVISFYNVTNKSHIFTFTHNFSGPLRPFFEPCLHDGGKNTAPLVICSELHKSEESIVPRPEGKGHANGDVSLKVNSSLLPPKAPELKDIILSLPPDLGPALQELKAPSF.

In an embodiment of the invention, the BTNL2 is human BTNL2. In anembodiment human ERMAP protein has the sequence:

(SEQ ID NO: 3) MVDCPRYSLSGVAASFLFVLLTIKHPDDFRVVGPNLPILAKVGEDALLTCQLLPKRTTAHMEVRWYRSDPAMPVIMYRDGAVVTGLPMEGYGGRAEWMEDSTEEGSVALKIRQVQPSDDGQYWCRFQEGDYWRETSVLLQVAALGSSPNIHVEGLGEGEVQLVCTSRGWFPEPEVHWEGIWGEKLMSFSENHVPGEDGLFYVEDTLMVRNDSVETISCFIYSHGLRETQEATIALSERLQTELVSVSVIGHSQPSPVQVGENIELTCHLSPQTDAQNLEVRWLRSRYYPAVHVYANGTHVAGEQMVEYKGRTSLVTDAIHEGKLTLQIHNARTSDEGQYRCLFGKDGVYQEARVDVQVTAVGSTPRITREVLKDGGMQLRCTSDGWFPRPHVQWRDRDGKTMPSFSEAFQQGSQELFQVETLLLVTNGSMVNVTCSISLPLGQEKTARFPLSDSKI.

In an embodiment of the invention, the short form of BTNL2 is a shortform of human BTNL2. In an embodiment the short form of human BTNL2 hasthe sequence:

(SEQ ID NO: 4) MVDCPRYSLSGVAASFLFVLLTIKHPDDFRVVGPNLPILAKVGEDALLTCQLLPKRTTAHMEVRWYRSDPAMPVIMYRDGAVVTGLPMEGYGGRAEWMEDSTEEGSVALKIRQVQPSDDGQYWCRFQEGDYWRETSVLLQVAALGSSPNIHVEGLGEGEVQLVCTSRGWFPEPEVHWEGIWGEKLMSFSENHVPGEDGLFYVEDTLMVRNDSVETISCFIYSHGLRETQEATIALSERLQ TELVSVSVIGHSQPSPVQVG.

Protein sequence of the IgV1 domain of human BTNL2:

(SEQ ID NO: 5) KQSEDFRVIGPAHPILAGVGEDALLTCQLLPKRTTMHVEVRWYRSEPSTPVFVHRDGVEVTEMQMEEYRGWVEWIENGIAKGNVALKIHNIQPSDNGQYWCHFQDGNYCGETSLLLKVAGLGSAPSIHM.

Protein sequence of the IgV1 domain of human BTNL9:

(SEQ ID NO: 6) EVKVLGPEYPILALVGEEVEFPCHLWPQLDAQQMEIRWFRSQTFNVVHLYQEQQELPGRQMPAFRNRTKLVKDDIAYGSVVLQLHSIIPSDKGTYGCRFHSDNFSGEALWELEVAGLGSDPHLS.

In an embodiment of the methods, the tumor is a tumor of a breast, lung,thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate,kidney, esophagus, or is a hematological tumor. In an embodiment of themethods, the tumor is a hematological tumor and is a leukemia or alymphoma. In an embodiment of the methods, the tumor is a tumor of thebreast and is a triple negative breast cancer. In an embodiment of themethods, the tumor is a tumor of a lymphoid organ.

Also provided is an isolated fusion protein comprising a soluble portionof an BTNL9 and an Fc portion of an immunoglobulin G.

Also provided is an isolated fusion protein comprising a soluble portionof an ERMAP and an Fc portion of an immunoglobulin G.

An isolated chimeric nucleic acid encoding an isolated fusion protein asdescribed herein is provided.

A composition comprising the isolated fusion protein as described hereinand a carrier is provided.

In an embodiment, the composition is a pharmaceutical composition, andthe carrier is a pharmaceutical carrier.

Also provided is a method of treating an autoimmune disease in a subjectcomprising administering to the subject an amount of an isolated,plasma-soluble BTNL9 to treat the autoimmune disease.

Also provided is a method of treating an autoimmune disease in a subjectcomprising administering to the subject an amount of an isolated,plasma-soluble ERMAP to treat the autoimmune disease.

Also provided is a method of treating an autoimmune disease in a subjectcomprising administering to the subject an amount of an isolated,plasma-soluble BTNL2 to treat the autoimmune disease.

In an embodiment, the BTNL-2 comprises SEQ ID NO:4 but does not compriseSEQ ID NO:3.

In an embodiment, the plasma-soluble BTNL9, or the plasma-soluble ERMAP,respectively, comprises an BTNL9 fused to an immunoglobulin polypeptide,or an ERMAP fused to an immunoglobulin polypeptide, respectively.

In an embodiment, the plasma-soluble BTNL2 comprises an BTNL2 fused toan immunoglobulin polypeptide.

In an embodiment, the immunoglobulin polypeptide comprises an Fc portionof an immunoglobulin G.

Also provided is an isolated, recombinant fusion polypeptide comprisinga BTNL9 fused to an immunoglobulin polypeptide. Also provided is anisolated, recombinant fusion polypeptide comprising an IgV1 domain ofhuman BTNL9 fused to an immunoglobulin polypeptide. In an embodiment,the IgV1 domain of human BTNL9 comprises SEQ ID NO:6.

Also provided is an isolated, recombinant fusion polypeptide comprisingan ERMAP fused to an immunoglobulin polypeptide.

Also provided is an isolated, recombinant fusion polypeptide comprisinga BTNL2 fused to an immunoglobulin polypeptide. In an embodiment, theBTNL-2 comprises SEQ ID NO:4 but does not comprise SEQ ID NO:3. Alsoprovided is an isolated, recombinant fusion polypeptide comprising anIgV1 domain of human BTNL2 fused to an immunoglobulin polypeptide. In anembodiment, the IgV1 domain of human BTNL2 comprises SEQ ID NO:5.

Also provided is an isolated chimeric nucleic acid encoding an isolatedrecombinant fusion polypeptide as described herein.

Also provided is a composition comprising the isolated recombinantfusion polypeptide as described herein and a carrier. In an embodiment,the compositions is a pharmaceutical composition, and comprises apharmaceutical carrier.

The term “ERMAP-Ig” fusion protein as used herein means a fusion proteinconstructed of a portion of an immunoglobulin and an active portion of aERMAP, or proteins having an identical sequence thereto. In a preferredembodiment, the active portion of a ERMAP is a soluble portion of ERMAP.In an embodiment, the ERMAP has the sequence of a human ERMAP. In anembodiment, the portion of an immunoglobulin is a portion of an IgG oran IgM. In an embodiment, it as a portion of an IgG. The IgG portion ofthe fusion protein can be, e.g., any of an IgG1, IgG2, IgG2a, IgG2b,IgG3 or IgG4 or a portion thereof. In an embodiment, the portion is anFc region. In an embodiment the fusion protein comprises a sequenceidentical to an Fc portion of a human IgG1, human IgG2, human IgG2a,human IgG2b, human IgG3 or human IgG4. In an embodiment the fusionprotein comprises a sequence identical to an Fc portion of a human IgG1.The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine of the Fc region may be removed, for example, byrecombinantly engineering the nucleic acid encoding the fusion protein.

The term “BTNL9-Ig” fusion protein as used herein means a fusion proteinconstructed of a portion of an immunoglobulin and an active portion of aBTNL9, or proteins having an identical sequence thereto. In anembodiment, the active portion of a BTNL9 is a soluble portion of BTNL9.In an embodiment, the BTNL9 has the sequence of a human BTNL9.

The term “BTNL2-Ig” fusion protein as used herein means a fusion proteinconstructed of a portion of an immunoglobulin and an active portion of aBTNL2, or proteins having an identical sequence thereto. In anembodiment, the active portion of a BTNL2 is a soluble portion of BTNL2.In an embodiment, the BTNL2 has the sequence of a human BTNL2. In anembodiment, the BTNL-2 comprises SEQ ID NO:4 but does not comprise SEQID NO:3.

In an embodiment, the portion of an immunoglobulin of the fusionproteins is a portion of an IgG or an IgM. In an embodiment, it as aportion of an IgG. The IgG portion of the fusion protein can be, e.g.,any of an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4 or a portion thereof.In an embodiment, the portion is an Fc region. In an embodiment thefusion protein comprises a sequence identical to an Fc portion of ahuman IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or humanIgG4. In an embodiment the fusion protein comprises a sequence identicalto an Fc portion of a human IgG1. The term “Fc region” herein is used todefine a C-terminal region of an immunoglobulin heavy chain, includingnative sequence Fc regions and variant Fc regions. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The C-terminal lysine of the Fc region may beremoved, for example, by recombinantly engineering the nucleic acidencoding the fusion protein.

In an embodiment, the Fc portion of the Ig is used in the fusionproteins as described herein. The presence of the Fc domain markedlyincreases the plasma half-life of the attached protein, which prolongstherapeutic activity. In addition, the Fc domain also enables the fusionprotein to interact with Fc-receptors. In an embodiment, the ERMAP-Igcomprises a ERMAP portion linked to an Fc domain. In an embodiment, theERMAP portion is bound directly by a peptide bond to the Fc domain. Inan embodiment, the ERMAP portion is linked to the Fc domain through alinker. In an embodiment, the BTNL9-Ig comprises a BTNL9 portion linkedto an Fc domain. In an embodiment, the BTNL9 portion is bound directlyby a peptide bond to the Fc domain. In an embodiment, the BTNL9 portionis linked to the Fc domain through a linker.

In an embodiment, the fusion protein (or fusion polypeptide) is linkedvia a peptide linker which permits flexibility. In an embodiment, thelinker is rigid. In an embodiment the linker is cleavable. Non-limitingexamples of flexible linkers within the scope of the invention areG_(n), and GGGGS, and (GGGGS)_(n) where n=2, 3, 4 or 5 (SEQ ID NO:7).Non-limiting examples of rigid linkers within the scope of the inventionare (EAAAK)_(n) (SEQ ID NO:8), (XP)_(n). Non-limiting examples ofcleavable linkers within the scope of the invention include disulfidelinks and protease cleavable linkers. In a preferred embodiment, thelinker is a peptide linker.

In an embodiment, the Fc domain has the same sequence or 95% or greatersequence similarity with a human IgG1 Fc domain. In an embodiment, theFc domain has the same sequence or 95% or greater sequence similaritywith a human IgG2 Fc domain. In an embodiment, the Fc domain has thesame sequence or 95% or greater sequence similarity with a human IgG3 Fcdomain. In an embodiment, the Fc domain has the same sequence or 95% orgreater sequence similarity with a human IgG4 Fc domain. In anembodiment, the Fc domain is not mutated. In an embodiment, the Fcdomain is mutated at the CH2-CH3 domain interface to increase theaffinity of IgG for FcRn at acidic but not neutral pH (Dall'Acqua et al,2006; Yeung et al, 2009).

In an embodiment, the fusion protein described herein is recombinantlyproduced. In an embodiment, the fusion protein is produced in aeukaryotic expression system. In an embodiment, the fusion proteinproduced in the eukaryotic expression system comprises glycosylation ata residue on the Fc portion corresponding to Asn297.

In an embodiment, the fusion protein is a homodimer. In an embodiment,the fusion protein is monomeric. In an embodiment, the fusion protein ispolymeric.

In an embodiment, a BTNL9-Ig is prepared by fusing the coding region ofthe extracellular domain of a BTNL9 having the same sequence as a humanextracellular domain of a BTNL9 to a polypeptide having the samesequence as a human IgG1 Fc. Such can be made in any way known in theart, including by transfecting an appropriate cell type with arecombinant nucleic acid encoding the fusion protein. The BTNL2-Igfusion protein can be made in an analogous matter, as can the otherfusion proteins mentioned herein.

In an embodiment, a ERMAP-Ig is prepared by fusing the coding region ofthe extracellular domain of a ERMAP having the same sequence as a humanextracellular domain of a ERMAP to a polypeptide having the samesequence as a human IgG1 Fc. Such can be made in any way known in theart, including by transfecting an appropriate cell type with arecombinant nucleic acid encoding the fusion protein.

In an embodiment, of all aspects of the invention described hereinreciting a subject, the subject is a human

Cancers, including tumors, treatable by the invention include of thenasopharynx, pharynx, lung, bone, brain, sialaden, stomach, esophagus,testes, ovary, uterus, endometrium, liver, small intestine, appendix,colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast,cervix, vagina, vulva, prostate, thyroid, skin, or is a glioma. In anembodiment, the cancer treated is a metastatic melanoma.

This invention also provides a composition comprising a fusion proteinas described herein. In an embodiment, the composition is apharmaceutical composition. In an embodiment the composition orpharmaceutical composition comprising one or more of the fusion proteinsdescribed herein is substantially pure with regard to the fusionprotein. A composition or pharmaceutical composition comprising one ormore of the fusion proteins described herein is “substantially pure”with regard to the antibody or fragment when at least about 60 to 75% ofa sample of the composition or pharmaceutical composition exhibits asingle species of the fusion protein. A substantially pure compositionor pharmaceutical composition comprising one or more of the fusionproteins described herein can comprise, in the portion thereof which isthe fusion protein, 60%, 70%, 80% or 90% of the fusion protein of thesingle species, more usually about 95%, and preferably over 99%. Fusionprotein purity or homogeneity may be tested by a number of means wellknown in the art, such as polyacrylamide gel electrophoresis or HPLC.

The invention also encompasses compositions comprising the describedfusion proteins and a carrier. The carrier may comprise one or morepharmaceutically-acceptable carrier components. Suchpharmaceutically-acceptable carrier components are widely known in theart.

In an embodiment, the subject being treated for cancer via a methodherein is also treated with a chemotherapeutic agents, such as acytotoxic agent. In an embodiment, the cytotoxic agent is doxorubicin.In an embodiment, the cytotoxic agent is a maytansinoid. In anembodiment, the cytotoxic agent an alkylating agent, an anti-metabolite,a plant alkaloid or terpenoid, or a cytotoxic antibiotic. Inembodiments, the cytotoxic agent is cyclophosphamide, bleomycin,etoposide, platinum agent (cisplatin), fluorouracil, vincristine,methotrexate, taxol, epirubicin, leucovorin (folinic acid), oririnotecan.

Administration as used herein, unless otherwise stated, can beauricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical,endosinusial, endotracheal, enteral, epidural, via hemodialysis,interstitial, intrabdominal, intraamniotic, intra-arterial,intra-articular, intrabiliary, intrabronchial, intrabursal,intracardiac, intracartilaginous, intracaudal, intracavernous,intracavitary, intracerebral, intracisternal, intracorneal,intracoronary, intradermal, intradiscal, intraductal, intraepidermal,intraesophagus, intragastric, intravaginal, intragingival, intraileal,intraluminal, intralesional, intralymphatic, intramedullary,intrameningeal, intramuscular, intraocular, intraovarian,intraepicardial, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous,intratesticular, intrathecal, intrathoracic, intratubular, intratumor,intratympanic, intrauterine, intravascular, intravenous,intraventricular, intravesical, intravitreal, laryngeal, nasal,nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous,periarticular, peridural, rectal, inhalationally, retrobulbar,subarachnoid, subconjuctival, sublingual, submucosal, topically,transdermal, transmucosal, transplacental, transtracheal, ureteral,uretheral, and vaginal.

In an embodiment, the fusion protein of the invention is administeredsystemically in the methods described herein. In an embodiment, thefusion protein of the invention is administered locally in the methodsdescribed herein. In an embodiment, the fusion protein of the inventionis administered directly to the tumor in the methods described herein,for example by injection or cannulation.

In an embodiment, the antibody or antibody fragment of the invention isadministered systemically in the methods described herein. In anembodiment, the antibody or antibody fragment of the invention isadministered locally in the methods described herein. In an embodiment,the antibody or antibody fragment of the invention is administereddirectly to the tumor in the methods described herein, for example byinjection or cannulation.

In an embodiment, “determining” as used herein means experimentallydetermining.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS

The existing technologies for T-cell based immunotherapies regardingB7/CD28 family members work by blockade of the B7/CD28 family members.In contrast, the expression patterns and functions of BTNL9 and ERMAP,two members of the butyrophilin family, are different from CTLA-4 andPD-1 and their ligands, therefore, the BTNL9 pathway and the ERMAPpathway regulate the immune system at the different times and locations.

Example 1

mRNA expression of BTNL9, ERMAP and other members of the butyrophilinfamily in tissues and antigen-presenting cells: The butyrophilin familyis related to the B7 family, but their expression and functions in theimmune system are largely unknown. Using Real-Time RT-PCR, it wasdetermined that BTNL9 and ERMAP, together with other family members,were widely expressed in many tissues and antigen-presenting cells (FIG.1).

Example 2

BTNL9, ERMAP and some other members of the butyrophilin family inhibit Tcell function: It was examined whether BTNL9, ERMAP and other members ofthe butyrophilin family were able to regulate T-cell function using asystem modified from previous studies (PNAS, 110: 9879-9884, 2013). Inthis system, purified T cells were activated with plate-bound mAb to CD3and the activation of T cells was determined on days 3 and Tcell-derived cytokines were determined on day 2. It was determined thatBTNL9 inhibited both CD4 and CD8 T cell proliferation and cytokineproduction, while ERMAP inhibited CD4, but not CD8 T cell proliferation(FIG. 2).

Example 3

Activated T cells have receptors for BTNL9, ERMAP and some other membersof the butyrophilin family: Because BTNL9 and ERMAP inhibited T cellfunction, it was examined if activated T cells express receptors forthese molecules. It was determined that BTNL9-Ig, BTNL9, ERMAP-Ig andother Ig fusion proteins of some members of the butyrophilin familybound to activated CD4 and CD8 T cells which were activated for threedays (FIG. 3).

Example 4

The IgV1 domain of Human BTNL2 is the functional domain. As shown inFIG. 4, the IgV1 domain of human BTNL2 bound human CD8 T cells, human Bcells, and human NK cells. BTNL2-IgV1-Ig protein (open histograms) andcontrol Ig (shaded histograms) are shown in FACS assays. Sequence shownis SEQ ID NO:5.

Example 5

The IgV1 domain of Human BTNL9 is the functional domain. As shown inFIG. 5, the IgV1 domain of human BTNL9 bound human CD8 T cells, human Bcells, and human NK cells. BTNL9-IgV1-Ig protein (open histograms) andcontrol Ig (shaded histograms) are shown in FACS assays. Sequence shownis SEQ ID NO:6.

1. A method of treating a tumor in a subject comprising administering tothe subject an amount of a BTNL9-binding antibody, or BTNL9-bindingfragment thereof, sufficient to inhibit a BTNL9 and treat the tumor. 2.A method of treating a tumor in a subject comprising administering tothe subject an amount of a ERMAP-binding antibody, or ERMAP-bindingfragment thereof, sufficient to inhibit a ERMAP and treat the tumor. 3.A method of treating a tumor in a subject comprising administering tothe subject an amount of a BTNL2-binding antibody, or BTNL2-bindingfragment thereof, sufficient to inhibit a BTNL2 and treat the tumor. 4.The method of claim 1, wherein the BTNL9 is a human BTNL9.
 5. The methodof claim 2, wherein the ERMAP is a human ERMAP.
 6. The method of claim3, wherein the BTNL2 is a human BTNL2.
 7. The method of claim 1, whereinthe tumor is a tumor of a breast, lung, thyroid, melanoma, pancreas,ovary, liver, bladder, colon, prostate, kidney, esophagus, or is ahematological tumor, or wherein the tumor is a lymphoid organ tumor. 8.The method of claim 1, wherein the antibody is administered as anadjunct to an additional anti-cancer therapy for the tumor.
 9. Themethod of claim 1, wherein the amount of a BTNL9-binding antibody isadministered.
 10. The method of claim 3, wherein the amount of aBTNL2-binding antibody is administered.
 11. The method of claim 1,wherein the fragment of the antibody is administered. 12-15. (canceled)16. The method of claim 15, wherein the BTNL-2 comprises SEQ ID NO:4 butdoes not comprise SEQ ID NO:3.
 17. The method of claim 13, wherein theplasma-soluble BTNL9, or the plasma-soluble ERMAP, respectively,comprises an BTNL9 fused to an immunoglobulin polypeptide, or an ERMAPfused to an immunoglobulin polypeptide, respectively.
 18. The method ofclaim 15, wherein the plasma-soluble BTNL2 comprises an BTNL2 fused toan immunoglobulin polypeptide.
 19. The method of claim 17, wherein theimmunoglobulin polypeptide comprises an Fc portion of an immunoglobulinG. 20-26. (canceled)